This invention relates to the field of therapeutics, particularly infections, in animals and humans. It relates to the design, synthesis and application of oligonucleotide analogs which mimic the RNA secondary structures found in diseased cells, particularly cells infected with viruses and retroviruses. These mimics of the infectious RNA structures have been found to be able to modulate such infections.
The biological function of RNA is mediated by its structure. mRNA is generally thought of as a linear molecule which contains the information for directing protein synthesis within the sequence of ribonucleotides. Recently, studies have revealed a number of secondary and tertiary structures in mRNA which are important for its function (See; I. Tinoco, P. W. Davis, C. C. Hardin, J. D. Puglisi, G. T. Walker, Cold. Spring. Harb. Symp. Quant. Biol. 52, 135 (1987). Secondary structural elements in RNA are formed largely by Watson-Crick type. interactions between different regions of the same RNA molecule. Important secondary structural elements include intramolecular double stranded regions, hairpin loops, bulges in duplex RNA and internal loops. Tertiary structural elements are formed when secondary structural elements come in contact with each other or with single stranded regions to produce a more complex, three dimensional structure.
Very little is known about the precise three dimensional structure of RNA. However, there have recently been a number of research efforts which have shown that RNA structures, including single stranded, secondary, and tertiary structures, have important biological functions beyond simply encoding information to make proteins in linear sequences. Some of these correlations have been discussed in: I. Tinoco, P. W. Davis, C. C. Hardin, J. D. Puglisi, G. T. Walker, Cold. Spring. Barb. Symp. Quant. Biol. 52, 135 (1987); O. Resnekov, M. Kessler, Y. Aloni, J. Bido. Chem. 264, 9953 (1989); C. Tuerk, P. Gauss, C. Thermes, et al, Proc. Natl. Acad. Sci. U. S. A. 85, 1364 (1988); D. E. Larson, B. H. Sells, Mol. Cell. Biochem. 74, 5 (1987); and G. Knapp, Methods Enzymol. 180, 192 (1989).
Oligonucleotides have been evaluated for effect on HIV. Agarwal and coworkers have used oligonucleotide analogs targeted to the splice donor/acceptor site to inhibit HIV infection in early infected and chronically infected cells. S. Agarwal, T. Ikeuchi, D. Sun, P. S. Sarin, A. Konopka, J. Maizel, Proc. Natl. Acad. Sci. USA 86:7790 (1989). Sarin and coworkers have also used chemically modified oligonucleotide analogs targeted to the cap and splice donor/acceptor sites. P. S. Sarin, S. Agarwal, M. P. Civerira, J. Goodchild, T. Ikeuchi, P. C. Zamecnik, Proc. Natl. Acad. Sci. USA 85:7448 (1988). Zaia and coworkers have also used an oligonucleotide analog targeted to a splice acceptor site to inhibit HIV. Zaia, J. A., J. J. Rossi, G. J. Murakawa, P. A. Spallone, D. A. Stephens, B. E. Kaplan, J. Virol. 62:3914 (1988). Matsukura and coworkers have synthesized oligonucleotide analogs targeted to the initiation of translation of the rev gene mRNA. M. Matsukura, K. Shinozuka, G. Zon, et al., Proc Natl. Acad. Sci. USA, 84:7706 (1987); R. L. Letsinger, G. R. Zhang, D. K. Sun, T. Ikeuchi, P. S. Sarin, Proc. Natl. Acad. Sci. USA 86:6553 (1989). Mori and coworkers have used a different oligonucleotide analog targeted to the same region as Matsukura et al., K. Mori, C. Boiziau, C. Cazenave et al., Nucleic Acids Res. 17:8207 (1989). Shibahara and coworkers have used oligonucleotide analogs targeted to a splice acceptor site as well as the reverse transcriptase primer binding site. S. Shibahara, S. Mukai, H. Morisawa, H. Nakashima, S. Kobayashi, N. Yamamoto, Nucl. Acids Res. 17:239 (1989). Letsinger and coworkers have synthesized and tested oligonucleotide analogs with conjugated cholesterol targeted to a splice site. K. Mori, C. Boiziau, C. Cazenave, et al., Nucleic Acids Res. 17:8207 (1989). Stevenson and Iversen have conjugated polylysine to oligonucleotide analogs targeted to the splice donor and the 5xe2x80x2-end of the first exon of the tat gene. M. Stevenson, P. L. Iversen, J. Gen. Virol. 70:2673 (1989). Each of these publications have reported some degree of success in inhibiting some function of the HIV virus. While each of these references is distinct from the approach of the present invention, each supports the view that nucleotide therapeutics in HIV infection is rational and based upon sound scientific principles. In each of these references the approach has been to design antisense oligonucleotides complementary to some portion of the HIV mRNA. The present invention relates to oligonucleotides which mimic an RNA and bind to a protein, rather than oligonucleotides which bind to the HIV RNA.
Heretofore, there have been no suggestions in the art of methods or materials which could be useful for mimicking the secondary or tertiary structures of RNA in order to modulate the expression of genes or to treat disease. This is despite the long-felt need for methods of therapeutics and for methods of inhibiting gene expression which may be related to diseases or disease states in animals. Accordingly, there remains a long-felt need for therapeutic materials and methods, especially for viruses and retroviruses.
It is a principal object of the invention to provide compositions and therapies for human diseases, particularly viral and retroviral infections.
It is a further object of the invention to provide therapeutic compositions which mimic the structure of a natural RNA.
Yet another object of this invention is to modulate gene expression in cells.
Yet another object of this invention is to provide therapies for human immunodeficiency virus infection.
These and other objects of this invention will become apparent from a review of the instant specification.
It has now been discovered that expression of genes may be modulated through the employment of compositions which are capable of RNA mimicry. The use of such RNA mimics can interfere with gene expression and, when that expression is implicated in the etiology of disease, lead to methods of therapeutics. In accordance with this invention, it has now been found that certain portions of RNA coded by genomic material can have secondary and even tertiary structure which plays a significant role in gene expression. It has now been found that the interaction of certain RNA""s, especially messenger RNA""s having secondary or tertiary structures, with proteins may be inhibited through the employment of oligonucleotides or oligonucleotide analogs which mimic at least a portion of the RNA. Such mimicry can interfere with the protein-RNA interaction and, through such interference, interfere with gene expression and the maintenance of disease states.
In accordance with preferred embodiments of the present invention, methods for modulating expression of a gene are provided comprising selecting a portion of RNA coded by the gene, which RNA is capable of interacting with one or more proteins. An oligonucleotide or oligonucleotide analog is then prepared in such a way as to mimic said portion of the RNA. Cells containing the gene are then contacted with the oligonucleotide or oligonucleotide analog to effect such modulation of expression. It will generally be the case that the gene is of an infectious organism, such as a virus or retrovirus. Preferably, the gene is from human immuno-deficiency virus.
In accordance with other preferred embodiments, the protein is produced by a second portion of RNA coded by the infectious organism such as a virus or retrovirus. In such a case, the interaction of the protein with the RNA portion selected, if permitted to occur, would generally effect stimulation of expression of the gene such that inhibition of this interaction effects repression or modulation of gene expression.
It is preferred that the oligonucleotide or oligonucleotide analogs of the invention mimic at least about 6 nucleotide units of the selected RNA. It is still more preferred that from 8 to about 60 nucleotide units be mimicked. From about 10 to about 30 nucleotide units are presently believed to be most preferred. In accordance with other preferred embodiments, the degree of mimicry of the selected RNA is such as to permit the oligonucleotide or oligonucleotide analog to achieve at least a portion of the secondary structure of the RNA.
In accordance with other preferred embodiments of the present invention, the TAR region, the CAR region, or the GAG-POL region of human immunodeficiency virus messenger RNA is targeted for oligonucleotide mimicry. The oligonucleotide or oligonucleotide analog is selected to be sufficient in its degree of mimicry as to be effective in interfering with the interaction of protein with the selected messenger RNA portions. Thus, for example, if the selected messenger RNA portion is the TAR region of HIV, then the oligonucleotide or oligonucleotide analog is constructed so as to mimic the TAR region sufficiently such that tat protein coded by another portion of the HIV messenger RNA is effectively complexed with or bound to the mimicking molecule. Similar considerations attend the preparation of oligonucleotide and oligonucleotide analog RNA mimics directed at the CAR and GAG-POL regions of HIV.